Pyrazoloquinolones as anticancer agents

ABSTRACT

Compounds of the formula ##STR1## wherein R 1  is hydrogen, lower-alkyl, or trifluoromethyl; 
     R 2  is lower-alkyl, trifluoromethyl or CH 2  Y where Y is hydroxy, chloro, lower-alkylamino or dilower-alkylamino 
     R 3  and R 4  are each independently hydrogen or fluoro; 
     R 5  is hydrogen, lower-alkyl or 
     (a) phenyl, 2-pyridyl, 4-pyridyl, 1-naphthyl or such groups substituted with one or more, the same or different, lower-alkyl, fluoro, hydroxy, or lower-alkoxy; 
     (b) cycloalkyl or cycloalkyl substituted with amino, lower-alkylamino, dilower-alkylamino, formamido, acetamido, lower-alkyl, hydroxy, lower-alkoxy, trifluoromethyl, carboxy, lower-alkoxycarbonyl or halo, or cycloalkyl having a phenyl ring fused thereto; 
     (c) A saturated heterocyclic 5- or 6-membered ring containing oxygen and/or nitrogen or such ring substituted with lower-alkyl; or 
     (d) (CH 2 ) n  -Z wherein n is an integer from about one to four and Z is amino, dilower-alkylamino, bis(hydroxylower-alkyl)lower-alkylamino; or a pharmaceutically acceptable acid addition salt thereof are topoiosmerase II inhibitors and are useful in the treatment of cancer in mammalian hosts.

BACKGROUND OF THE INVENTION a) Field of the Invention

This invention relates to novel pyrazolo [4,3-c]quinolin-3-ones, and topharmaceutical compositions of these compounds and a method of usethereof as anticancer agents.

b) Information Disclosure Statement

Yokoyama, U.S. Pat. No. 4,312,870, issued Jan. 26, 1982, discloses(2-aryl-pyrazolo[4-3-]quinolin-3-ones of formula: ##STR2## wherein R isphenyl, R"-phenyl, pyridyl, alkylpyridyl or halopyridyl;

R" is hydrogen, alkyl, alkoxy, alkylthio, OH, halo, CF₃, nitro, amino,mono-or dialkylamino, CN, carbamoyl or carboxy; and

pharmaceutically acceptable acyl derivatives or salts thereof, which arestated to be psychoactive agents .

Yokoyama, U.S. Pat. No. 4,524,146, issued Jun. 18, 1985, disclosespyrazolo[4,3-c]quinolon-3-ones of the formula ##STR3## wherein R₁ is anaromatic heterocyclic radical selected from quinolyl, isoquinolyl,pyrimidyl and thiazolyl, or such said heterocyclic radical mono- ordi-substituted lower alkyl-, lower alkoxy or halogen;

R₂ and R₃ each independently represents hydrogen or lower alkyl, and

R₄ and R₅ each independently represent hydrogen, lower alkyl,lower-alkoxy, halogen or trifluoromethyl; and

pharmaceutically acceptable salts thereof, which are stated to bebenzodiazepine receptor modulators.

Yokoyama, U.S. Pat. No. 4,602,014, issued Jul. 22, 1985, disclosescompound of the formula ##STR4## wherein A is an optionally substitutedsaturated divalent grouping which together with the two carbon atoms towhich is is attached represents a fused 5-, 6-, or 7-memberedcarbocyclic or heterocyclic ring selected from optionally substitutedfused cyclopenteno, cyclohexeno, cyclohepteno, dihydrothieno,dihydropyrano, tetrahydrooxepino, dihydropyrrolo, tetrahydropyrido andtetrahydroazepino;

R₁ is lower alkyl, phenyl, or phenyl mono- or disubstituted by loweralkyl, lower alkoxy, halogen or trifluoromethyl; or

R₁ is an aromatic heterocyclic radical selected from e.g. optionallysubstituted pyridyl, quinolyl, isoquinolyl, pyrimidyl and thiazolyl;

R₂, R₃ and R₃, are hydrogen or lower alkyl; and pharmaceuticallyacceptable salts are useful as benzodiazepine receptor modulators forthe treatment of nervous system disorders. Pharmaceutical compositions,methods of preparation and certain intermediates stated to be useful asbenzodiazepine receptor modulators are also disclosed.

International Patent Application Publication No. W091/06298, publishedMay 16, 1991, discloses the use of2,5-dihydro-2-phenyl-3H-pyrazolo(4,3c) quinolin -3-one or itspharmaceutically acceptable alkali metal or acid salts for treatinginflammatory and allergic diseases.

Wentland, U.S. Pat. No. 4,959,363, issued Sep. 25, 1990, disclosescompounds of the formula ##STR5## wherein R is hydrogen, hydroxy, aminoor lower-alkyl;

R₁ is lower-alkyl, lower-alkenyl, cycloalkyl, pyridinyl, phenyl orsubstituted phenyl

R₂ is hydrogen, amino or hydroxy;

R₆ is hydrogen or fluoro; and

R₇ is phenyl, pyridinyl or selected other heterocycles;

which are stated to have antiviral activity against herpes virus.

Lesher et al., U.S. Pat. No. 5,075,319, dated Dec. 24, 1991, from anapplication filed Sep. 13, 1989, discloses fluorinated 1-cyclopropyl-7-(substituted-pyridinyl)-1,4-dihydro-4-oxo-3-quinolinecarboxylic acids ofthe formula ##STR6## wherein R is hydrogen;

R' and R" are hydrogen or fluoro, or other groups; and

Z is 3- or 4-pyridinyl substituted by alkyl groups or substituted alkylgroups. The compounds are stated to be superior antibacterial agents.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to novel 2-R₅-5-cyclopropyl-8-fluoro-6-R₄ -9-R₃ -2,5-dihydro-7-(2-R₂ -6-R₁-4-pyridinyl)-3H-pyrazolo-[4,3-c]-quinolin-3-ones useful asantineoplastic agents.

In a second aspect, the invention also relates to a pharmaceuticalcomposition containing as an active ingredient an antineoplasticeffective amount of a compound of the invention.

In a third aspect, the invention relates to a method of inhibiting thegrowth of or killing malignant cells which comprises administering to amammal afflicted with malignant cells a compound of the invention in anantineoplastic effective amount to inhibit the growth of or induce theregression of these cells.

DETAILED DESCRIPTION INCLUSIVE OF PREFERRED EMBODIMENTS

As used herein, the term lower-alkyl refers to a straight or branchedalkyl radical of 1 to about 6 carbon atoms including, for example,methyl, propyl, isopropyl, sec-butyl, pentyl, n-butyl, 3-hexyl and thelike. The term lower-alkoxy refers to a straight or branched alkoxyradical with from 1 to about 4 carbon atoms such as methoxy, ethoxy,propoxy, isopropoxy, butoxy, sec-butoxy and the like.

Cycloalkyl refers to a cyclic hydrocarbon radical of about 4 to about 7carbon atoms, for example, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and the like. Cycloalkyl having a phenyl ring fused theretois exemplified by 1,2,3,4-tetrahydronaphthyl, 2,3-dihydroindenyl and thelike.

As used herein the term halo or halogen refers to the common halogens,for example, fluoro, chloro, bromo or iodo.

Saturated heterocyclic 5- or 6-membered rings containing oxygen and/ornitrogen include, for example, pyrrolidinyl, morpholinyl, piperidinyl,and the like. These saturated heterocyclic radicals may be N- orC-substituted with lower-alkyl. When bonded directly to thepyrazoloquinolone, the point of attachment of the 5- or 6-memberedsaturated heterocycle is a ring carbon atom thereof. When bonded to thepyrazoloquinolone through an alkylene group (as Z in the group (CH₂)_(n)Z), the point of attachment of the heterocycle can be either a ringcarbon or nitrogen atom thereof.

Specifically, the present invention relates to compounds of formula I##STR7## wherein R₁ is hydrogen, lower-alkyl, or trifluoromethyl;

R₂ is lower-alkyl, trifluoromethyl or CH₂ Y where Y is hydroxy, chloro,lower-alkylamino or dilower-alkylamino

R₃ and R₄ are each independently hydrogen or fluoro;

R₅ is hydrogen, lower-alkyl or

(a) phenyl, 2-pyridyl,4-pyridyl, 1-naphthyl or such groups substitutedwith one or more, the same or different, lower-alkyl, fluoro, hydroxy,or lower-alkoxy;

(b) cycloalkyl or cycloalkyl substituted with amino, lower-alkylamino,dilower-alkylamino, formamido, acetamido, lower-alkyl, hydroxy,lower-alkoxy, trifluoromethyl, carboxy, lower-alkoxycarbonyl or halo, orcycloalkyl having a phenyl ring fused thereto;

(c) A saturated heterocyclic 5- or 6-membered ring containing oxygenand/or nitrogen or such ring substituted with lower-alkyl; or

(d) (CH₂)_(n) -Z wherein n is an integer from about one to four and Z isamino, dilower-alkylamino, bis(hydroxylower-alkyl)lower-alkylamino,hydroxy, trifluoromethyl, carboxy, lower-alkoxycarbonyl, lower-alkoxy,formyl or acetal thereof, or a substituent selected from (a), (b) or (c); or a pharmaceutically acceptable acid addition salt thereof.

Preferred compounds of formula 1 are those wherein R₁ and R₂ are methyl,R₃ is hydrogen, R₄ is fluoro and R₅ is as defined above.

Compounds of formula I may be prepared according to the followingscheme; ##STR8##

As the scheme illustrates, quinoline carboxylic acid II, described inU.S. Pat. No. 5,075,319, which is incorporated herein by reference, isprotected by esterification using methods well known in the art; forexample, the acid and lower-alkanol, such as methanol or ethanol, can berefluxed with 1', 1 carbonyldiimidazole, or in the presence of acatalytic amount of a strong acid, e.g. sulfuric acid.

In a preferred method, this protected quinolone III is then thionylatedusing, for example,[2,4-bis-(4-methoxyphenyl)-1,3-dithia-2,4-diphosphotane-2,4-disulfide](Lawesson's reagent), P₄ S₁₀ and the like, in an inert solvent betweenambient temperature and the boiling point of the solvent, yielding anintermediate of formula IV.

This intermediate is then reacted with an appropriate hydrazine, orsubstituted hydrazine (R₅ NHNH₂) in an inert solvent, for exampleacetonitrile or DMF, at a temperature between ambient and the boilingpoint of the solvent, preferably in the presence of a catalytic amountof base, for example pyridine and the like, yielding a compound offormula I.

The hydrazines, R₅ NHNH₂, are generally known and are commerciallyavailable or are prepared by methods well known in the art.

Simple chemical transformations which are conventional and well known tothose skilled in the art of chemistry can be used for effecting changesin functional groups in the compounds of the invention. For example,acylation of hydroxy- or amino-substituted species to prepare thecorresponding esters or amides, respectively; cleavage of methyl orbenzyl ethers to produce the corresponding alcohols or phenols; andhydrolysis of esters or amides to produce the corresponding acids,alcohols or amines as desired can be carried out.

The compounds of the invention are useful both in the free base form andthe form of acid-addition salts, and both forms are within the purviewof the invention. The acid-addition salts are in some cases a moreconvenient form for use, and in practice the use of the salt forminherently amounts to the use of the base form. The acids which can beused to prepare the acid-addition salts include preferably those whichproduce, when combined with the free base, medicinally acceptable salts,that is, salts whose anions are relatively innocuous to the animalorganism in medicinal doses of the salts so that the beneficialproperties inherent in the free base are not vitiated by side effectsascribable to the anion. In practicing the present invention, it isconvenient to form the hydrochloride, fumarate, toluenesulfonate,hydrogen sulfate, methanesulfonate or maleate salts and the like.However, other appropriate medicinally acceptable salts within the scopeof the invention are those derived from other mineral acids and organicacids. The acid-addition salts of the basic compounds are preparedeither by dissolveing the free base in aqueous alcohol solutioncontaining the appropriate acid and isolating the salt by evaporatingthe solution, or by reacting the free base and an acid in an organicsolvent, in which case the salt separates directly, is precipitated witha second organic solvent, or can be obtained by concentration of thesolution. Although medicinally acceptable salts of the basic compoundsare preferred, all acid-addition salts are within the scope of thepresent invention. All acid-addition salts are useful as sources of thefree base form even if the particular salt per se is desired only as anintermediate product, as, for example, when the salt is formed only forpurposes of purification or identification, or when it is used as anintermediate in preparing a medicinally acceptable salt by ion exchangeprocedures.

The structures of the compounds of the invention were established by themode of synthesis, by elemental analysis, and by infrared, nuclearmagnetic resonance, and mass spectroscopy. The course of the reactionsand the identity and homogeneity of the products were assessed by thinlayer chromatography (TLC) and high-pressure liquid chromatography(HPLC).

The following examples illustrate the invention, but do not limit itthereto.

In the following example preparations, diethyl ether is referred to asether. All reactions were run in dried solvents and under a nitrogenatmosphere.

PREPARATION OF INTERMEDIATES Preparation 1

Ethyl 1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl) -4-oxo-3-quinolinecarboxylic acid Formula III R₁ =R₂ =CH₃, R₃=hydrogen, R₄ =fluoro (Intermediate 1)

1,1'-Carbonyldiimidiazole (CDI) (3.9 g, 0.024 mol) was added to asuspension of (6.0 g, 0.016 mol) 1-cyclopropyl-7-(2,6-dimethyl-4-pyridinyl )-6,8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid, described inU.S. Pat. No. 5,075,319, and incorporated herein by reference, in DMF(40 mL) and the mixture was stirred at 100° C. for 2 hr under a N₂atmosphere. The resulting solution was cooled to 25° C., diluted withand 60 mL ethanol and heated at reflux 17 h. Removal of the solvents invacuo gave a residue that was stirred in 20 mL ethanol; after cooling inice a solid separated and was collected (5.5 g, 86%); a 4.5 g portion ofthis material was recrystallized from ethanol to give 4.25 of ethyl1-cyclopropyl-7-(2,6-dimethyl-4-pyridinyl)-6,8-difluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylate,mp 193°-195° C.

Preparation 2

Ethyl 1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-thioxo-3-quinolinecarboxylate (Formula IV R₁ =R₂ =CH₃, R₃ =hydrogen,R₄ =fluoro) (Intermediate 2)

Lawesson's reagent (25.2 g, 0.06 mol) was added to a solution of ethyl1-cyclopropyl-7-(2,6-dimethyl-4-pyridinyl)-6,8-difluoro-1,4-dihydro-4-oxo -3-quinolinecarboxyate (24.1 g, 0.06 mol)in 720 mL THF and the mixture heated at reflux for 21 h. The solvent wasremoved in vacuo and the residue purified by column chromatography(silica gel 97:3/CHCl₃ :2-propylamine) to give 26.1 g of product.Recrystallization from ethyl acetate gave ethyl1-cyclopropyl-7-(2,6-dimethyl-4-pyridinyl)-6,8-difluoro-1,4-dihydro-4-thioxo-3-quinolinecarboxylate (21.0 g, 84%); mp194°-195° C.

EXAMPLE 1 (METHOD A)

5-Cyclopropyl-6,8-difluoro-2,5-dihydro-2-[2-(dimethylamino)-ethyl]-7-(2,6-dimethyl-4-pyridinyl )-3H-pyrazolo[4,3-c]quinolin-3-one (E)-2-butendioate (1:1).2-(Dimethylamino)ethyl hydrazine (7.7 g, 0.0186 mol) was added to asolution of Intermediate 2 (5.0 g, ) 0.045 mol) in 46 mL DMF and theresulting solution was heated at 100° C. for 3 h. The mixture was cooledin ice and a solid was collected and washed with ether to give 5.3 g(65%) of product which was treated with fumaric acid (2.72 g, 0.0234mol) in ethanol to give 6.7 g (54%) of the (E)-2-butendioate salt of acompound of formula I [R₁ =R₂ =CH₃, R₃ =hydrogen, R₄ =fluoro, R₅ =(CH₂)₂N(CH₃)₂ ], mp 224°-226° C.

EXAMPLE 2 (METHOD B)

4-Cyclopropyl-6,8-difluoro-2,5-dihydro-2-(2,2,2-trifluoroethyl)-7-(2,6-dimethyl-4-pyridinyl)-3H-pyrazolo[4,3-c]quinolin-3-one.A solution of Intermediate 2 (0.31 g, 0.75 mmol), 2,2,2-trifluoroethylhydrazine (0.33 g, 2.19 mmol) and 3 mL pyridine was heated at 100° C.for 2 h. An additional 0.30 g of 2,2,2-trifluoroethyl hydrazine wasadded and heating was continued for 10 h. The resulting mixture wasconcentrated and purified using silica gel chromatography to provide0.27 g (79%) of a compound of formula I [R₁ =R₂ =CH₃, R₃ =hydrogen, R₄=fluoro, R₅ =CH₂ CF₃ ], mp 255°-257° C.

EXAMPLE 3 (METHOD C)

5-Cyclopropyl-6,8-difluoro-7-(2,6-dimethyl-4-pyridinyl)-2,5-dihydro-2-(2-propyl) -3H-pyrazolo[4,3-c]quinolin-3-one. A mixture ofIntermediate 2 (0.50 g, 0.0012 mol), isopropyl hydrazine.2CH₃ CO₂ H(1.88 g, 0.006 mol) and 4-dimethylaminopyridine (0.88 g, 0.0072 mol) washeated at 120° C. for 2 h and cooled. The resulting mixture waspartitioned between CHCl₃ and water; the organic layer was washed withbrine, dried over Na₂ SO₄ and concentrated in vacuo. The crude productwas purified using silica gel chromatography to give 0.11 g (23%) of acompound of formula I [R₁ =R₂ =CH₃, R₃ =hydrogen, R₄ =fluoro, R₅=CH(CH₃)₂ ], mp 202°-204° C.

EXAMPLE 4 (METHOD D)

2-(1-Butyl)-5-cyclopropyl-6,8-difluoro-2,5-dihydro-7-(2,6-dimethyl-4-pyridinyl)-3H-pyrazolo[4,3-c]quinolin-3-one. A mixture of Intermediate 2 (0.70 g,0.0017 mol) n-butyl hydrazinc oxalate (0.80 g, 0.0045 mol),triethylamine (1.0 mL) and 4 mL DMF was heated at 100° C. for 7 h. Themixture was cooled and concentrated and the crude product was purifiedusing silica gel chromatography to give 0.30 g (43%) of a compound offormula I [R₁ =R₂ =CH₃, R₃ =hydrogen, R₄ =fluoro, R₅ =(CH₂)₃ CH₃ ]; mp164°-165° C.

EXAMPLE 5 (METHOD E)

5-Cyclopropyl-6,8-difluoro-2,5-dihydro-2-(2-hydroxyphenyl)-7-(2,6-dimethyl-4-pyridinyl)-3H-pyrazolo[4,3-c]quinolin-3-one. A solution of Example 32 (0.24 g,0.51 mmol) in 6 mL CH₂ Cl₂ at -78° C. was treated with BBr₃ (0.20 mL).After stirring at -78° C. for 15 min and 25° C. for 90 min, the mixturewas treated with 5 mL of 10% NaOH and diluted with water. To thismixture methylene chloride was added followed by 4 mL acetic acid. TheCH₂ Cl₂ layer was dried over Na₂ SO₄ and concentrated. The crude productwas purified by silica gel chromatography to give 0.19 g (83%) of acompound of formula I [R₁ =R₂ =CH₃, R₃ =hydrogen, R₄ =fluoro, R₅=2-HO-C₆ H₄ ], mp 232°-233° C.

EXAMPLES 6-55

The following compounds of formula I wherein R₁ =R₂ =CH₃, R₃ =hydrogen,R₄ =fluoro, were prepared from Intermediate 2 by reaction with anappropriate hydrazine, R₅ NHNH₂, substantially as described in examples1-4, (methods A-D respectively), and methoxy-containing compounds offormula I were converted to the corresponding hydroxy compounds asdescribed in Example 5 (Method E). In the table, recrystallizingsolvents are abbreviated as follows: ethyl acetate (EA), methylenechloride (CH₂ Cl₂), hexanes (hex), isopropanol (i-PrOH), ethanol (EtOH)and diethyl ether (Et₂ O). In cases where no recrystallizing solvent isindicated recrystallization was not carried out. Melting points are ofthe free base unless otherwise specified. The notation (d) refers todecomposition at the temperature listed, and (S) indicates that themelting point listed is that of the methanesulfonate salt prepared bythe procedure used in method A, substituting methane sulfonic acid forfumaric acid. The hydrazines used are commercially available, known orprepared by known methods.

                                      TABLE I                                     __________________________________________________________________________    Compounds of Formula I (R.sub.1 ═R.sub.2 ═CH.sub.3, R.sub.3           ═H, R.sub.4 ═F                                                                                              recrystalizing                          Example                                                                            R.sub.5         mp (°C.)                                                                       method                                                                            yld (%)                                                                            solvent                                 __________________________________________________________________________     6   H               >310    A   97                                            7   CH.sub.3        242-243 A   35                                            8   C(CH.sub.3).sub.3                                                                             219-221 D   48   EA                                       9   CH.sub.2 CO.sub.2 Et                                                                          204-206 D   64                                           10   CH.sub.2 CH.sub.2 OH                                                                          274-276 A   67                                           11   (CH.sub.2).sub.4 OH                                                                           233-234 B   72   CH.sub.2 Cl.sub.2 :hex                  12   CH.sub.2 CH(OCH.sub.3).sub.2                                                                  172-173 B   57                                           13   (CH.sub.2).sub.2 CN                                                                           268-270 B   43                                           14   (CH.sub.2).sub.3 N(CH.sub.3).sub.2                                                            169-171 A   27   EA                                      15   (CH.sub.2).sub.2 N(CH.sub.2 CH.sub.3).sub.2                                                     212-216 (S)                                                                         B   54                                           16   (CH.sub.2).sub.3 N(CH.sub.2 CH.sub.3).sub.2                                                   195-197 B   39                                           17   (CH.sub.2).sub.2 NH(CH.sub.2).sub.2 OH                                                        158 (d)(S)                                                                            B   34   iPrOH                                   18   (CH.sub.2).sub.2 -1-pyrrolidinyl                                                              248-250 B   54                                           19   (CH.sub.2).sub.2 -1-piperidinyl                                                               234 (d)(S)                                                                            B   49                                           20   (CH.sub.2).sub.2 -4-(morpholino)                                                              264-265 B   95   Acetone                                 21   (CH.sub.2).sub.2 2-(1-CH.sub.3)pyrrolidinyl                                                   203-205 B   33                                           22   C.sub.6 H.sub.5 210 (d) A   31   EtOH                                    23   4-CH.sub.3 OC.sub.6 H.sub.4                                                                   212-215 A   21   EA                                      24   4-CH.sub.3 C.sub.6 H.sub.4                                                                    194-196 A   33                                           25   C.sub.6 F.sub.5 198-214 A   49                                           26   2-CH.sub.3 OC.sub.6 H.sub.4                                                                   200 (d) B   70                                           27   4-HOC.sub.6 H.sub.4                                                                           >300    E   71   DMF                                     28   1-naphthyl      280-282 B   42                                           29   2-pyridinyl     259-261 B   63                                           30   4-pyridinyl     287-289 B   30   Acetone                                 31   CH.sub.2 C.sub.6 H.sub.5                                                                      243-245 D   47                                           32   CH.sub.2 -2-CH.sub.3 OC.sub.6 H.sub.4                                                         213-215 B   58   Et.sub.2 O                              33   CH.sub.2 -2-HOC.sub.6 H.sub.4                                                                 265-269 E   45                                           34   CH.sub.2 -4-CH.sub.3 OC.sub.6 H.sub.4                                                         196-198 B   70                                           35   CH.sub.2 -4-HOC.sub.6 H.sub.4                                                                 302-303 E   56                                           36   CH.sub.2 -3,4,5-(CH.sub.3 O).sub.3 C.sub.6 H.sub.2                                            228-230 B   86                                           37   (CH.sub.2).sub.2 C.sub.6 H.sub.5                                                              221-223 B   72                                           38   (CH.sub.2).sub.2 -2-CH.sub.3 OC.sub.6 H.sub.4                                                 230-232 B   100                                          39   (CH.sub.2).sub.2 -2-HOC.sub.6 H.sub.4                                                         >300    E   50                                           40   cyclohexyl      259-261 D   52                                           41   cyclopentyl     259-260 B   83                                           42   cycloheptyl     248-250 B   47                                           43   CH.sub.2 -cyclohexyl                                                                          190-193 C   31   Et.sub.2 O                              44   1,2,3,4-tetrahydro-2-naphthyl                                                                 225-235 B   52   Acetone                                 45   cis-2-CH.sub.3 O-cyclohexyl                                                                   210-212 B   62                                           46   4-tetrahydropyranyl                                                                           282-284 B   86                                           47   4-CH.sub.3 -cyclohexyl                                                                        200-204 B   51                                           48   4-C.sub.6 H.sub.5 -cyclohexyl                                                                 152-207 B   44                                           49   4-piperidinyl   240 (d) B   80                                           50   1-CH.sub.3 -4-piperidinyl                                                                     218 (d) B   62                                            51a (cis)4-(CH.sub.3).sub.2 N-cyclohexyl*                                                         137-140 B   14                                            51b (trans)4-(CH.sub.3).sub.2 N-cyclohexyl*                                                       156-159 B   26                                            52a (cis)4-CH.sub.3 CONH-cyclohexyl*                                                              282 (d) C    5                                            52b (trans)4-CH.sub.3 CONH-cyclohexyl*                                                            245 (d) C    3                                           __________________________________________________________________________     *Isomers separated by chromatography                                     

EXAMPLE 53

2-[5-Cyclopropyl-6,8-difluoro-2,5-dihydro-7-(2,6-dimethyl-4-pyridinyl)-3-oxo-3H-pyrazolo[4,3-c]quinolinyl]aceticacid. A mixture of example compound 9 (0.29 g, 0.00064 mol), acetic acid(3.2 mL), water (0.8 mL), and 12N HCl (0.8 mL) was heated at 100° C. for3 h. After concentration, the residue was taken up in DMF, chilled, andfiltered. The filtrate was concentrated and the resulting solid wasslurried in THF. The solid was collected and washed with ether to give0.21 g (73%) of a compound of formula I [R₁ =R₂ =CH₃, R₃ =hydrogen, R₄=fluoro, R₅ =CH₂ COOH], mp 200° C.(d).

EXAMPLE 54

5-Cyclopropyl-6,8-difluoro-2,5-dihydro-2-[4-(dimethylamino)-butyl]-7-(2,6-dimethyl-4-pyridinyl) -3H-pyrazolo[4,3-c]quinolin-3-one. p-Toluenesulfonylchloride (0.24 g, 1.25 mol) was added to a mixture of example compound11 (Formula I R₁ =R₂ =CH₃, R₃ =hydrogen, R₄ =fluoro, R₅ =(CH₂)₄ OH)(0.42 g, 0.96 mmol) and 15 mL pyridine. The mixture was heated at 50° C.for 3 h and additional p-toluenesulfonyl chloride (0.050 g) was addedfollowed by continued heating for another 1 h. The mixture was pouredinto 1N HCl and was washed with ether. The aqueous portion was basifiedwith 1N NaOH to pH 7.8 and was extracted with CH₂ Cl₂. After drying (Na₂SO₄) the extracts were concentrated to give 0.26 g of crude rosylatewhich was dissolved in 15 mL ethanol saturated with dimethylamine. Theresulting mixture was heated in a stainless steel pressure vessel at110° C. for 2 hours. The reaction mixture was cooled and concentrated togive a crude product that was purified by silica gel chromatography togive 0.143 g (35%) of a compound of formula I [R₁ =R₂ =CH₃, R₃=hydrogen, R₄ =fluoro. R₅ =(CH₂)₄ N(CH₃)₂ ], mp 173°-175° C.

EXAMPLE 55

2-[[2-(2,2-Bis(hydroxymethyl)ethylamino]ethyl]-5-cyclopropyl-6,8-difluoro-2,5-dihydro-7-(2,6-dimethyl-4-pyridinyl)-3H-pyrazolo[4,3-c]quinolin-3-one. To a stirred suspension of example compound 10 (FormulaI R₁ =R₂ =CH₃, R₃ =hydrogen, R₄ =fluoro, R₅ =(CH₂)₂ OH) (1.326 g, 3.23mmol) in pyridine (7 mL) was added p-toluenesulfonyl chloride (0.74 g,3.9 mmol). After warming on a steam bath at 60° C. for 75 min, the clearsolution was evaporated on a rotary evaporator to leave asemi-crystalline solid (2.53 g) . To this crude tosylate was addeddiisopropylethylamine (10 mL and 2-amino-2-methyl-1,3-propanediol (11.4g) . The mixture was stirred at reflux under N₂ for 15 min cooled. Theupper mobile liquid layer was decanted and the remaining hard glassymaterial was taken up in warm water (75 mL). The solution was filtered,then extracted with CH₂ Cl₂ (2×50 mL). The extracts were washed withwater, dried (MgSO₄) and evaporated to afford a golden-yellow solid(0.544 g). Purification of the crude product was effected by silica gelchromatography to give a compound of formula I [R₁ =R₂ =CH₃, R₃=hydrogen, R₄ =fluoro, R₅ =(CH₂)₂ NHC(CH₂ OH)₂ CH₃ ](0.106 g, 6.4%) asan amorphous solid.

EXAMPLE 56

2-(Cis-4-Aminocyclohexyl)-5-cyclopropyl-6,8-difluoro-2,5-dihydro-7-(2,6-dimethyl-4-pyridinyl)-3H-pyrazolo-[4,3-c]quinolin-3-one(56a) and 2-(trans-4-aminocyclohexyl)-5-cyclopropyl-6,8-difluoro-2,5dihydro-7-(2,6-dimethyl-4-pyridinyl)-3H-pyrazolo[4,3-c]quinolin-3-one(56b). The unseparated isomers .of example 52 (0.3 g, 0.59 mmol) and 2NHCl (27 mL) were heated at reflux for 14 h. The resulting solution wasbasified with Na₂ CO₃ and extracted with CH₂ Cl₂. The organic portionwas dried (Na₂ CO₃) and concentrated to give a crude product containingthe isomeric amines. Purification of each isomer was done by silica gelchromatography to give 0.10 g (38%) of the cis isomer, a compound offormula I [R₁ =R₂ =CH₃, R₃ =hydrogen, R₄ =fluoro, R₅ =(cis) 4-aminocyclohexyl], example 56a, mp 260°-260° C.; and 0.070 g (25%) of thetrans isomer, example 56b, a compound of formula I [R₁ =R₂ =CH₃, R₃=hydrogen, R₄ =fluoro, R₅ =(trans) 4-amino cyclohexyl], mp 225°-228° C.

EXAMPLE 57

2-(cis-4-formamidocyclohexyl)-5-cyclopropyl-6,8-difluoro-2,5-dihydro-7-(2,6-dimethyl-4-pyridinyl)-3H-pyrazolo[4,3-c]quinolin-3-one A solution containingexample compound 56a (0.144 g, 0.31 mmol) and N,N-dimethylformamidedimethyl acetal (10 mL) was heated at 50° C. for 13 h and concentrated.The crude product was purified using silica gel chromatography to give0.124 g (77%) of a compound of formula I (R₁ =R₂ =CH₃, R₃ =hydrogen, R₄=fluoro, R₅ =(cis) 4-formamidocyclohexyl), mp 250° C. (decomposed).

EXAMPLE 58

2-(trans-4-formamidocyclohexyl)-5-cyclopropyl-6,8-difluoro-2,5-dihydro-7-(2,6-dimethyl-4-pyridinyl)-3H-pyrazolo[4,3-c]quinolin-3-oneA solution containing example compound 56b (0.092 g, 0.00012 mol) andN,N-dimethylformamide dimethyl acetal (10 mL) was heated at 50° C. for10 h and concentrated. The crude product was purified using silica gelchromatography to give 0.53 g (53%) of a compound of formula I [R₁ =R₂=CH₃, R₃ =hydrogen, R₄ =fluoro, R₅ =(trans) 4-formamido-cyclohexyl), mp165°-167° C.

It is contemplated that the following intermediates of formula II, asdescribed in U.S. Pat. No. 5,075,319, which is incorporated herein byreference, when esterified and thionylated as described in preparations1 and 2 above, yield corresponding intermediates of formula IV;

1-cyclopropyl-6-fluoro-1,4-dihydro-7-(2,6-di(trifluoromethyl)-4-pyridinyl)-4-oxo-3-quinolinecarboxylic acid,

1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-(2-ethyl,6-hydroxymethyl)-4-pyridinyl)-4-oxo-3-quinoline carboxylic acid,

1-cyclopropyl-5,6,8-trifluoro-1,4-dihydro-7-(2-methyl,6-methoxymethyl-4-pyridinyl)-4-oxo-3-quinolinone carboxylic acid,

1-cyclopropyl-6-fluoro-1,4-dihydro-7-(2-ethyl,6-chloromethyl)-4-pyridinyl)-4-oxo-3-quinoline carboxylic acid, and

1-cyclopropyl-6-fluoro-1,4-dihydro-7-(2-isopropyl,6-methylaminomethyl-4-pyridinyl)-4-oxo-3-quinoline carboxylic acid.

It is further contemplated that compounds of formula I are prepared fromany of these intermediates by condensation with any of the examplehydrazines, using methods A-D above.

BIOLOGICAL PROPERTIES

Topoisomerase II has been identified as the cellular target for a numberof therapeutically important antineoplastic classes of drugs (Glissonand Ross, Pharmacol. Ther. 32, 89-106, 1987; Liu, Ann. Rev. Biochem. 58,351-375, 1989). These chemically distinct agents include intercalatinganthracyclines, aminoacridines, and ellipticines as well as the non-DNAintercalating epipodophyllotoxins. The intracellular effects of theseagents (Zwelling et al., Biochem, 20, 6553-6563, 1981; Long et al,,Biochem. 23, 1183-1188, 1984; Rowe et al. Biochem. Pharmacol. 34,2483-2487, 1985; Rowe et al., Cancer Res. 46, 2021-2026, 1986; Kerriganet al., NCI Monographs 4:117-121, 1987; Covey et al., Cancer Res. 48,860-865, 1988), in addition to their topoisomerase II reactivity invitro (Nelson et al., Proc. Natl. Acazd, Sci. 81, 1361-1365, 1984; Teweyet al., J. Biol. Chem. 259, 9182-9187, 1984a; Tewey et al., Science 266,466-468, 1984b; Ross et al., Cancer Res. 44, 5857-5860, 1984; Chen etal., J. Biol. Chem. 259, 13560-13566, 1984; Rowe et al., Cancer Res. 46,2021-2026, 1986), implicate topoisomerase II inhibition as central tothe cytotoxicity and antitumor activity of these antineoplastic agents.Additionally the mechanisms of resistance observed in severalantineoplastic agent resistant cell lines appears to be a consequence ofeither an alteration in the topoisomerase enzyme molecule (Pommier etal., Cancer Res. 46, 3075-308a, 1986; Glisson et al., Cancer Res. 46,1934-1938, 1986; Esty et al., Biochem. Biophys. Res. Commun. 144,787-793, 1987; Danks et al., Biochem. 27, 8861-8869, 1988; Sinha et al.,Cancer Res. 48, 5096-5100, 1988) or its level (Per et al., Mol.Pharmacol. 32, 17-25, 1987). This evidence has clearly establishedtopoisomerase II inhibition as a means of deriving an antitumor effect.

The compounds of the present invention are inhibitors of mammaliantopoisomerase II, thus indicating their use as cytotoxic andantineoplastic agents in the chemotherapy of cancer in mammals.

MAMMALIAN TOPOISOMERASE II INHIBITION ASSAY PROCEDURE

The inhibition of human topoisomerase II (hereafter topo II) wasquantitated by a procedure adapted from that described by Trask et al.,EMBO J., 3, 671-676 (1984). The assay quantitates the amount of topo IIcovalently attached to DNA at equilibrium during a topo II reaction.This assay determines the potential of a compound to stabilize thiscomplex, which potential is closely related to the cytotoxicity of thecompound.

Topo II was purified from late log phase suspension cultures of HeLa WISby an adaptation of the method described by Per et al., Mol. Pharmacol.,32, 17-25 (1987).

Assays (in duplicate) were assembled at 4° C. Assay mix (25 μl) wasdistributed in Beckman (No. 265270) 1.5 mL microtiter tubes followed bythe addition of 5 μl test compound to yield the final concentrations ofassay components:

50 mM Tris-Cl pH 7.9

44 mM NaCl

10 mM MgCl₂

0.6 mM DTT

0.5 mM EDTA

30 μg/ml BSA

0.5 mM ATP

5.5% (w/v)glycerol

0.4 ng 3' end labeled (³² p) pBR322 DNA (10⁷ DPM/μg)

10 units Topo II

The assay mix including the test compound was incubated for 20 minutesat 37° C. The reaction was terminated at 37° C. by the addition of 3 μl10% SDS followed by the addition of 266 μl 10 mM Tris-Cl pH 7.5, 20μg/ml BSA, 20 μg/ml calf thymus DNA, 1% SDS.

A SDS/protein precipitate was formed by the addition of 28 μl 2.5M KClfollowed by chilling on ice for a minimum of 10 minutes. The precipitatewas collected and washed with a Brandell cell harvester on a GFB glassfiber filter membrane as follows. The contents of the assay tube weredrawn up into the harvester. The tube was then rinsed 7× with 10 mMTris-Cl pH 7.5, 1 mM EDTA and 100 mM KCl. The precipitate was washedwith 1 L of a solution of 10 mM Tris-Cl pH 7.5, 1 mMEDTA, 100 mM KClfollowed by 1 L of 95% ethyl alcohol and finally 0.5 L 70% of ethylalcohol (per 48 samples in each case). After drying, CPM was determinedby liquid scintillation counting with 5 ml Biofluor (NEN ResearchProducts) or Readisafe (Beckman Instruments Inc.) cocktail.

Preparation of test compound--A stock solution (6 mg/ml) of testcompound was prepared either in 0.1N sodium hydroxide or 0.2N hydrogenchloride. This solution then was diluted 1/5 into water and seriallythereafter in either 0.02N sodium hydroxide or 0.04N hydrogen chloride,respectively. The stock solution and serial dilution of the testcompound was stored at -20° C. prior to testing.

Screening of test compound--As an initial screen, the test compound wastested at a final concentration of 2, 20 and 200 μg/ml. The compound wasthen retested at a range of concentrations (usually 2-3×steps) bridgingtheir approximate EC₅₀ 's, as estimated by the prescreen.

Controls--A solvent control which indicates the base level of topoII-DNA complex formed in the absence of the test compound was includedin each test. A control, in which topo II was omitted, was included foreach test compound at the highest drug concentration tested.

Reference Agent--A dose response curve with mAMSA at 0.01, 0.08, 0.16,0.32, 1.0 and 10 μg/ml was included in each test.

Data reduction--The EC₅₀ (effective concentration at which 50% of themaximal DNA-topo II complex is formed) of a test compound is defined tobe the concentration with activity equal to the EC₅₀ of the referenceagent, mAMSA. The maximal DNA-topo II complex formed is taken as thatequal to that formed at the nearly saturating dose of mAMSA (10 μg/ml).

The results obtained for representative compounds of the invention inthe human topoisomerase II assay procedure expressed as EC₅₀ s (μM) arepresented in Table II below.

MAMMALIAN CANCER CELL CYTOTOXICITY ASSAYS

In vitro cytotoxicity was assayed by determining cell survival followingexposure of P388 murine leukemia cells to a range of test compoundconcentrations. Cytotoxicity was quantitated following either a one-hourexposure or continuous exposure of P388 cells to test compound.

Cytotoxicity following a one-hour exposure of cells to drug was assayedby quantitating clonogenic cell survival according to a publishedprocedure [Freshney, R. L. (1987) Culture of Animal Cells. A Manual ofBasic Technique. Wiley-Liss Inc., New York, NY, pp. 140-144]. Briefly,following drug exposure cells were plated in soft agar and incubated(37°, 5% CO₂) for 7-10 days during which time viable cells form visiblecolonies. Cytotoxicity following continuous exposure of cells to testcompound was assayed by quantitating total P388 cells on a CoultierCounter following a 48-hour co-incubation of test compound with cells.P388 cells were maintained in log phase throughout the duration ofcytotoxicity assays.

The percentage of cells surviving relative to an untreated control wasgraphed as a function of increasing drug concentration. The IC₅₀ isdefined as that concentration of drug which reduced the population ofviable cells to 50% that of an untreated control.

                  TABLE II                                                        ______________________________________                                        Topoisomerase Inhibition and In Vitro Cytotoxicity of                         Representative Examples of Compounds of Formula I                                    topo II inh..sup.a                                                                         in vitro cytotoxicity.sup.b                               Example  EC.sub.50 - μM                                                                            1 hr exp.                                                                              cont. exp.                                   ______________________________________                                         1       2.6.sup.c      0.26     0.04                                          2       6.8            --       1.4                                           3       5.0            --       0.53                                          4       2.9            --       1.5                                           5       1.2            --       0.15                                          6       11             7.9      3.6                                           7       5.9            --       0.51                                          8       6.9            --       3.5                                           9       6.1            --       0.15                                         10       6.4            --       1.4                                          11       4.2            --       0.88                                         12       23             --       --                                           13       6.7            --       1.4                                          14       1.7            0.16     0.38                                         15       93.sup.c,d     0.45     --                                           16       6.0            0.49     1.2                                          17       3.3.sup.c      1.3      1.2                                          18       650.sup.c,d    0.3      0.035                                        19       210.sup.c,d    0.44     --                                           20       48             2.8      0.41                                         21       3.5            0.33     0.16                                         22       2.7            --       0.84                                         23       6.8.sup.c      --       5.2                                          24       8.9            --       4.9                                          25       3.4            --       2.2                                          26       5.0            --       0.34                                         27       4.9            --       2.6                                          28       2.9            --       0.32                                         29       2.8            --       0.74                                         30       8.0.sup.c      --       1.2                                          31       3.7            --       0.14                                         32       5.1            --       1.2                                          33       3.8            --       1.4                                          34       3.2            --       0.44                                         35       1.5            --       0.25                                         36       6.4            --       1.2                                          37       5.0            --       2.2                                          38       11             --       --                                           39       9.6.sup.c      --       --                                           40       0.90           0.68     0.19                                         41       4.0            1.1      0.38                                         42       2.9            4.2      1.5                                          43       8.2            6.7      4.9                                          44       3.7            --       1.5                                          45       6.3            0.57     --                                           46       1.7            0.29     0.25                                         47       3.1            1.4      --                                           48       4.5            8.4      --                                           49       5.6            2.3      2.5                                          50       3.2            0.094    --                                            51a     1.7            0.067    --                                            51b     4.4            0.26     --                                            52a     7.4            1.3      --                                            52b     9.8            1.2      --                                           53       10             --       18                                           54       15             0.40     0.50                                         55       11             11       25                                            56a     0.5            0.44     --                                            56b     4.2            1.5      --                                           57       0.80           5.1      --                                           58       1.5            9.1      --                                           ______________________________________                                         .sup. a Effective concentration that achieves 50% of the maximal effect o     mAMSA vs topo II isolated from HeLa cells (see experimental section).         .sup.b Inhibitory concentration vs. P388  IC50  μM (see Biological         Properties).                                                                  .sup.c Bellshaped dose response curve was noted when determining              IC.sub.50.                                                                    .sup.d Extrapolated IC.sub.50 value  50% inhibition was not observed at       the highest concentration of drug tested.                                

Representative examples of the compounds of formula I were also testedfor antitumor activity in mice against several tumor systems, asdescribed more fully below, and were found to possess antineoplasticactivity as evidenced by their activity in reducing the size of andcuring tumors, and increasing the survival time of the mice.

IN VIVO ANTITUMOR ASSAY PROCEDURE

Mice: Inbred: C3H/He and NCR-nu; and Hybrids: B6D2F1 (C57BL/6females×DBA/2 males, CD2F1 (Balb/c females×DBS/2 males) and B6C3fl(C57BL/6×C3H) were bred at Wayne State University from strains obtainedfrom the Frederick Cancer Research Facility, Frederick, Maryland orpurchased from commercial suppliers.

Tumors: Murine Tumor: P388 and an adriamycin-resistant (ADR) sublineP388/ADR leukemia and the following transplantable solid tumors of micewere used for in vivo testing: B16 melanoma (B16), pancreatic ductaladenocarcinoma No. 03 (Pane 03), colon adenocarcinoma No. 38 (colo 38),lung carcinoma No. 12 (LC 12), mammary ductal adenocarcinoma No. 16/C(Mam16C) and an adriamycin-resistant subline Mam16C/ADR. Human tumor: Asingle human tumor, mammary carcinoma MX-1 was used for in vivo testing.All tumors are in the Developmental Therapeutics Program frozen tumorrespository, maintained by the Biological Testing Branch, FrederickMaryland. Each has a detailed description, code identification number,and list of references at the National Tumor Repository. Murine tumorswere maintained in the mouse strain of origin and were transplanted inthe appropriate F1 hydrid (or the strain of origin) for therapy trials.Human mammary carcinoma MX-1 (MX1) was maintained as a subcutaneousimplant in either athymic Swiss (Cr: NIH(S)-nu) or athymic random bred(NCR-nu) mice and transplanted in NCR-nu for therapy trials.

Chemotherapy: For pancreatic ductal adenocarcinoma No. 3, colonadenocarcinoma No. 38 (colo 38), lung carcinoma No. 12 LC 12) and bothadriamycin-sensitive (RP) and-resistant (ADR) mammary ductaladenocarcinoma No. 16 (Mam16) tumors, bilateral tumor implants were usedto help ensure a more uniform tumor burden per mouse (thus reducing therequirement for greater numbers of mice per group). The animalsnecessary to begin an experiment were pooled, implanted bilaterally s.c.on day zero with 30-60 mg tumor fragments using a 12-gauge trocar, andagain pooled before randomization to the various treatment and controlgroups. Chemotherapy was started within three days after tumorimplantation while the number of cells per mouse was relatively small(1×10⁷ -1×10⁸ cells).

For P388 and P388/ADR leukemia studies the tumor cells were implantedeither interperitoneally (IP) or intravenously (IV) on day zero andtreatment was started on day one. For B16 melanoma (B16) studies, thetumor cells were implanted IP on day zero and treatment was started onday one. Titered controls were also included to facilitate thecalculation of tumor cell kill. For mammary carcinoma MX-1 studies,tumors were implanted with 1) subcutaneously (sc) (14-mg fragment of scdonor tumor) in the axillary region or 2) under the subrenal capsule(SRC) (10×10×10 ocular micrometer unit fragment of sc donor tumor).Treatment started on the day after subrenal capsule tumor implant orwhen the subcutaneous tumor implant had reached 100-700 mg.

END POINTS FOR ASSESSING ANTITUMOR ACTIVITY

Quantitative end points used to assess antitumor activity included %Increased Life Span (% ILS), Tumor Cell Kill (Log₁₀ kill), and TumorGrowth Inhibition (T/C). Long Term Survivors (45 or 60 day) wereexcluded from calculations of %ILS and Tumor Cell Kill.

Endpoints were calculated as follows: ##EQU1## where D_(t) is the medianday of death for treated and D_(c) is the median day of death forcontrol groups. A % ILS ≧20 or ≧25 for P388 and B16 intraperitonealmodels, respectively, is indicative of a significant degree of antitumoractivity. A % ILS ≧75 or ≧50 for P388 and B16 intraperitoneal models, ,respectively, is indicative of a high degree of antitumor activity andis the level used by National Cancer Institute to justify furtherdevelopment if other requirements are met (termed DN-2 level activity).Minimum quantitative activity limits for additional P388 models (e.g.P388/ADR and P388, IV) have not been defined. However, the activitylimits specified for the intraperitoneal models may be applied as in themajority of instances the intraperitoneal models are found to besignficantly less challenging.

TUMOR CELL KILL

The log₁₀ cell kill was calculated from the following formula: ##EQU2##where T-C is the tumor difference in the median day of death between thetreated (T) and the control (C) groups and Td is the tumor doubling time(in days), the latter estimated from the best fit straight line from alog-linear growth plot of the control-group tumors in exponentialgrowth. The conversion of the T-C values to log₁₀ cell kill is possiblebecause the Td for tumors regrowing post-treatment approximated the Tdvalues of the tumors in untreated control mice.

T/C VALUE

Tumors were measured with a caliper once or twice weekly (as needed)until either tumors exceeded 1600 mg or cure was assured. Tumor weightswere estimated from two-dimensional measurements: Tumor Weight(mg)=(a×b²) / 2, where a and b are the tumor length and width (ram)respectively. Measurements were carried out simultaneously in bothtreatment and control groups. When the control group tumors reachedapproximately 750-1500 mg in size (median of group), the median tumorweight of each group was determined (including zeros). The T/C value inpercent is an indication of antitumor effectiveness. The % T/C wascalculated from the following formula for solid murine tumor models:##EQU3## where T and C are median tumor weights of the treatment andcontrol groups, respectively. A T/C equal to or less than 42% isconsidered significant antitumor activity. A T/C value <10% isindicative of a high degree of antitumor activity and is the level usedby National Cancer Institute to justify further development if otherrequirements are met (termed DN-2 level activity). By convention the T/Cvalue for the mammary carcinoma MX-1 models is calculated by theparameter of change in tumor weight . The % T/C was calculated from thefollowing formula for MX-1 models ##EQU4## where ΔT and ΔC are thechange in mean tumor weight of the test and control groups,respectively, and T (initial) is the initial mean tumor weight of thetest group. An initial % T/C ≦20 is considered necessary to demonstratemoderate activity. A reproducible % T/C ≦10 is considered significantactivity.

ACTIVITY

In vivo trials of representative examples of the compounds of formula Iare summarized in Table III.

                                      TABLE III                                   __________________________________________________________________________    In vivo Properties of Representative Examples of Compounds of Formula I                        Drug       MTD          Log                                  Example                                                                            Exp Model   Route                                                                             Schedule                                                                             Mg/Kg                                                                              T/C %                                                                             ILS %                                                                             Kill                                 __________________________________________________________________________     1   SD-17                                                                             B16(ip) ip  qd 1,5,9                                                                             78       85  1.9                                       1038                                                                              Colo38(sc)                                                                            iv, sc                                                                            iv; qd 3-16                                                                          58   0       1.2                                       1038                                                                              Colo38(sc)                                                                            iv  qd 3,7,11                                                                            78   14      0.60                                      1161                                                                              LC12(sc)                                                                              iv  qd 3,5 35.sup.1                                                                           25      0.76                                      924 Mam16C/RP                                                                             iv  bid 1-5                                                                              20   13      1.2                                           (sc)                                                                      1120A                                                                             Mam16C/ADR                                                                            iv  qd 1,3,5                                                                             58.sup.2                                                                           26      0.50                                      1132                                                                              Mam16C(sc)                                                                            iv  qd 1-4 43   7       1.3                                       1142A                                                                             Mam16C(sc)                                                                            iv  qd 1,3,5                                                                             43   18      1.9                                       SD-9                                                                              MXI(sc) ip  qd 1,5,9                                                                             78   37                                                SD-18                                                                             MXI(src)                                                                              ip  qd 1,5,9                                                                             52   6                                                 1073B                                                                             P388/ADR(iv)                                                                          iv  qd 1-5 48       14.3                                                                              0.93                                      1149B                                                                             P388/ADR(iv)                                                                          iv  qd 1-3 52       27  1.3                                       1155A                                                                             P388(iv)                                                                              iv  qd 1-3 37       82  5.4                                       SD-7                                                                              P388(ip)                                                                              iv  qd 1,5,9                                                                             117      170 12                                        SD-7                                                                              P388(ip)                                                                              ip  qd 1,5,9                                                                             78       155 11                                        895A                                                                              Panc03(sc)                                                                            iv  qd 3   35   32      0.66                                      910 Panc03(sc)                                                                            iv  qd 3-9 45   45                                                1005A                                                                             Panc03(sc)                                                                            iv  qd 3,4 52   66                                                1146                                                                              Panc03(sc)                                                                            iv  qd 3,6,9                                                                             34   8       0.66                                  6   783 Panc03(sc)                                                                            sc  qd 16-18                                                                             2500.sup.1                                                                         >100                                                              qd 3-13                                                  14   SD-17                                                                             B16(ip) ip  qd 1,5,9                                                                             28       58.sup.3                                                                          1.3                                       905 Colo38(sc)                                                                            iv  qd 3,12-14                                                                           133  38      0.54                                      924 Mam16C/RP                                                                             iv  bid 1-8                                                                              101  8       1.5                                       SD-9                                                                              MXI(sc) ip  qd 1,5,9                                                                             42   53                                                SD-7                                                                              P388(ip)                                                                              iv  qd 1,5,9                                                                             64       70  5.0                                       SD-7                                                                              P388(ip)                                                                              ip  qd 1,5,9                                                                             42       85  6.1                                       910 Panc03(sc)                                                                            iv  bid 3-13                                                                             107  16                                                895A                                                                              Panc03(sc)                                                                            iv  qd 3-5 64   35      0.60                                 17   SD-17                                                                             B16(ip) ip  qd 1,5,9                                                                             180      61  1.4                                       SD-9                                                                              MXI(sc) ip  qd 1,5,9                                                                             270  67                                                SD-7                                                                              P388(ip)                                                                              iv  qd 1,5,9                                                                             120      5   0.35                                      SD-7                                                                              P388(ip)                                                                              ip  qd 1,5,9                                                                             180      50  3.5                                       988 Panc03(sc)                                                                            iv,sc                                                                             iv; qd 3,9-11                                                                        1410 42                                                                bid 4-5,12-15                                                                 sc; bid 16,18                                                                 qd 17,19                                                 21   1097                                                                              Panc03(sc)                                                                            iv  qd 3-4 80   35                                           22   861 Panc03(sc)                                                                            sc  qd 3-7 635  >100                                         23   887B                                                                              panc03(sc)                                                                            sc  qd3-8  1150 18                                                953 Panc03(sc)                                                                            iv  (3-hr continu-                                                                       140  18                                                                ous infusion)                                            50   1086                                                                              Panc03(sc)                                                                            iv  qd 3-14                                                                              128  24                                            51a SD-17                                                                             B16(ip) ip  qd 1,5,9                                                                             15.sup.1 29.sup.2,3                                                                        0.67                                      1036                                                                              Colo38(sc)                                                                            iv  qd 3-4 63   39                                                                bid 5,6,12-17                                                 SD-18                                                                             MXI(src)                                                                              ip  qd 1,5,9                                                                             23.sup.1                                                                           5                                                 SD-15                                                                             P388(ip)                                                                              iv  qd 1,5,9                                                                             39.9     70  5.1                                       SD-16                                                                             P388(ip)                                                                              ip  qd 1,5,9                                                                             9.9      65  4.7                                       1005A                                                                             Panc03(sc)                                                                            iv  qd 3-4,8-12                                                                          63   5                                             51b SD-17                                                                             B16(ip) ip  qd 1,5,9                                                                             99.sup.1 47.sup.2,3                                                                        1.0                                       1036                                                                              Colo38(sc)                                                                            iv  qd 3-4,15,17                                                                         220.sup.1                                                                          13.sup.1                                                          bid 5.7                                                       SD-18                                                                             MXI(src)                                                                              ip  qd 1,5,9                                                                             99   2                                                 SD-16                                                                             P388(ip)                                                                              iv  qd 1,5,9                                                                             120      85  6.2                                       SD-16                                                                             P388(ip)                                                                              ip  qd 1,5,9                                                                             66       85  6.2                                       1005A                                                                             Panc03(sc)                                                                            iv,sc                                                                             iv; qd 3-4,                                                                          390.sup.1                                                                          2                                                                 8-14                                                                          sc; bid 15,                                               56a 1163                                                                              Colo38(sc)                                                                            iv,sc                                                                             iv; qd 3-9                                                                           137  40                                                                sc; qd 11-19                                                  1086                                                                              Panc03(sc)                                                                            iv  qd 3-4, 8-14                                                                         110  21                                            56b 1086                                                                              Panc03(sc)                                                                            iv,sc                                                                             iv; qd 3-6                                                                           550  42                                                                bid 7-12                                                                      sc; qd 15                                                                     bid 13-14                                                __________________________________________________________________________     .sup.1 MTD not achievedmaximum dose tested.                                   .sup.2 2 lower doses equally active to modestly more active.                  .sup.3 1/6 tumor free long term survivor.                                

In practicing the method of the invention, the therapeutic dose of thecompound of formula I to be administered to the mammal afflicted withmalignant cells is that amount which is effective to inhibit mammaliantopoisomerase II and thereby to inhibit the growth of, kill or inducethe regression of the malignant cells, or to prolong the life of themammal.

The specific amount of formula I constituting a therapeuticallyeffective dose and the length of treatment required will vary since itis dependent on a number of factors such as, for example, the size, age,condition and species of the mammal to be treated, the degree ofinvolvement of the malignancy, the specific compound to be administeredand its bioavailability, the dose regimen and the mode ofadministration. The specific amount to be employed for a particularafflicted mammal is readily determinable by the skilled artisan usingconventional techniques.

In practicing the invention, the compounds can be administered to themammal orally or parenterally.

The compounds can be prepared for use by incorporating them inconventional, pharmaceutically acceptable diluents, carriers orexcipients. For parenteral administration (intravenous, intraperitoneal,subcutaneous or intramuscular), the compounds are dissolved or suspendedin an aqueous or nonaqueous vehicle. For oral administration, thecompounds are formulated in dosage unit form as tablets or capsules.Exemplary diluents, carriers or excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,mineral oil, cocoa butter, alginates, tragacanth, gelatin, methylcellulose, methyl- and propyl hydroxybenzoates, talc, magnesium stearateand the like.

I claim:
 1. A compound of formula ##STR9## wherein R₁ is hydrogen,lower-alkyl, or trifluoromethyl;R₂ is lower-alkyl, trifluoromethyl orCH₂ Y where Y is hydroxy, chloro, lower-alkylamino or dilower-alkylaminoR₃ and R₄ are each independently hydrogen or fluoro; R₅ is hydrogen,lower-alkyl or(a) phenyl, 2-pyridyl, 4-pyridyl, 1-naphthyl or suchgroups substituted with one to about three, the same or different,lower-alkyl, fluoro, hydroxy, or lower-alkoxy; (b) C₄ to C₇ cycloalkylor C₄ to C₇ cycloalkyl substituted with amino, lower-alkylamino,dilower-alkylamino, formamido, acetamido, lower-alkyl, hydroxy,lower-alkoxy, trifluoromethyl, carboxy, lower-alkoxycarbonyl or halo, orC₄ to C₇ cycloalkyl having a phenyl ring fused thereto, forming a C₈ toC₁₁ bicyclic radical wherein 1 ring is aromatic and the other saturated;(c) A saturated carbon containing heterocyclic 5- or 6-membered ringcontaining oxygen and/or nitrogen or such ring substituted withlower-alkyl where if both nitrogen and oxygen are present they arenon-adjacent; or (d) (CH₂)_(n) -Z wherein n is an integer from one tofour and Z is amino, dilower-alkylamino, bis(hydroxylower-alkyl)lower-alkylamino, hydroxy, trifluoromethyl, carboxy,lower-alkoxycarbonyl, lower-alkoxy, formyl or acetal thereof, or asubstituent selected from (a), (b) or (c); or a pharmaceuticallyacceptable acid addition salt thereof.
 2. A compound according to claim1 wherein R₁ and R₂ are methyl, R₃ is hydrogen, and R₄ is fluoro.
 3. Acompound according to claim 2 wherein R₅ is hydrogen, 2-pyridyl,4-pyridyl, phenyl, 1-naphthyl or phenyl substituted with one or more offluoro, methyl, methoxy or hydroxy.
 4. A compound according to claim 2wherein R₅ is a cyclopentyl, cyclohexyl, cycloheptyl, or1,2,3,4-tetrahydro-2-naphthyl, or cyclohexyl substituted withdimethylamino, amino, acetamido or formamido.
 5. A compound according toclaim 2 wherein R₅ is piperidinyl, tetrahydropyranyl, pyrrolidinyl orpiperidinyl substituted with methyl.
 6. A compound according to claim 2wherein R₅ is (CH₂)_(n) Z, n is 1-3 and Z is phenyl, phenyl substitutedin the 2 or 4 position with methoxy or hydroxy, 3,4,5(CH₃ O)₃ phenyl,ethoxy carbonyl, bis (hydroxymethyl) ethylamino, hydroxy, diethylamino,dimethylamino, 2-hydroxyethylamino, cyano, cyclohexyl, acetamido,tetrahydropyranyl, 1-pyrrolidinyl, 1-piperidinyl, 4-morpholino or2-(1-CH₃)-pyrrolidinyl.
 7. A compound according to claim 2 wherein R₅ ishydrogen, phenyl, cyclohexyl, 2(N,N-dimethylamino)ethyl,3(N,N-dimethylamino)propyl, 2-(N-hydroxyethylamino)ethyl,2-(1-methyl)-2-pyrrolidinyl)ethyl, 4(N,N-dimethylamino)-cyclohexyl,4-aminocyclohexyl, 4-(N-methylamino)cyclohexyl.
 8. A compound accordingto claim 6 having the formula ##STR10##
 9. A pharmaceutical compositioncomprising a carrier and as an active ingredient a compound according toclaim 1 in a sufficient amount to inhibit the growth of malignant cellssusceptable to the action of such compound.
 10. A pharmaceuticalcomposition comprising a carrier and as an active ingredient a compoundaccording to claim 2 in a sufficient amount to inhibit the growth ofmalignant cells susceptable to the action of such compound.
 11. Apharmaceutical composition comprising a carrier and as an activeingredient a compound according to claim 3 in a sufficient amount toinhibit the growth of malignant cells susceptable to the action of suchcompound.
 12. A pharmaceutical composition comprising a carrier and asan active ingredient a compound according to claim 4 in a sufficientamount to inhibit the growth of malignant cells susceptable to theaction of such compound.
 13. A pharmaceutical composition comprising acarrier and as an active ingredient a compound according to claim 5 in asufficient amount to inhibit the growth of malignant cells susceptableto the action of such compound.
 14. A pharmaceutical compositioncomprising a carrier and as an active ingredient a compound according toclaim 6 in a sufficient amount to inhibit the growth of malignant cellssusceptable to the action of such compound.
 15. A pharmaceuticalcomposition comprising a carrier and as an active ingredient a compoundaccording to claim 7 in a sufficient amount to inhibit the growth ofmalignant cells susceptable to the action of such compound.
 16. Apharmaceutical composition comprising a carrier and as an activeingredient a compound according to claim 8 in a sufficient amount toinhibit the growth of malignant cells susceptable to the action of suchcompound.